U.S. patent application number 12/933669 was filed with the patent office on 2011-04-21 for injectable sustained-release pharmaceutical formulation and the preparation method thereof.
This patent application is currently assigned to Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, People Liberation Ar. Invention is credited to Junlin He, Qiyan Jia, Sicheng Li, Yuanjun Liang, Keliang Liu, Qingbin Meng, Dongqin Quan, Chenhong Wang.
Application Number | 20110091420 12/933669 |
Document ID | / |
Family ID | 41090463 |
Filed Date | 2011-04-21 |
United States Patent
Application |
20110091420 |
Kind Code |
A1 |
Liu; Keliang ; et
al. |
April 21, 2011 |
Injectable Sustained-Release Pharmaceutical Formulation and the
Preparation Method Thereof
Abstract
Disclosed are an injectable sustained-release pharmaceutical
formulation and a process for preparing the same. In some
embodiments, the formulation comprises an active ingredient in a
therapeutically effective amount, an amphipathic molecule, an
organic acid and/or a salt thereof which is hardly soluble in
water, and an oily solvent. The injectable sustained-release
pharmaceutical formulation provides a good sustained-release effect
for various active ingredients, in particular peptides, proteins,
nucleic acids and saccharides.
Inventors: |
Liu; Keliang; (Beijing,
CN) ; Quan; Dongqin; (Beijing, CN) ; Liang;
Yuanjun; (Beijing, CN) ; Meng; Qingbin;
(Beijing, CN) ; Wang; Chenhong; (Beijing, CN)
; He; Junlin; (Beijing, CN) ; Jia; Qiyan;
(Beijing, CN) ; Li; Sicheng; (Chengdu,
CN) |
Assignee: |
Institute of Pharmacology and
Toxicology, Academy of Military Medical Sciences, People Liberation
Ar
Beijing
CN
Chengdu Yiping Pharmaceutical Science Development Co.,
Ltd.
Chengdu (Sichuan)
CN
|
Family ID: |
41090463 |
Appl. No.: |
12/933669 |
Filed: |
March 20, 2009 |
PCT Filed: |
March 20, 2009 |
PCT NO: |
PCT/CN09/70913 |
371 Date: |
December 13, 2010 |
Current U.S.
Class: |
424/85.4 ;
514/11.1; 514/11.9; 514/15.2; 514/21.9; 514/282; 514/397; 514/44R;
514/5.9; 514/6.9; 514/9.7 |
Current CPC
Class: |
A61K 47/44 20130101;
A61K 47/02 20130101; A61K 9/127 20130101; A61K 9/0019 20130101;
A61K 47/12 20130101; A61K 47/24 20130101 |
Class at
Publication: |
424/85.4 ;
514/397; 514/282; 514/44.R; 514/21.9; 514/11.1; 514/11.9; 514/5.9;
514/15.2; 514/6.9; 514/9.7 |
International
Class: |
A61K 38/21 20060101
A61K038/21; A61K 31/415 20060101 A61K031/415; A61K 31/44 20060101
A61K031/44; A61K 48/00 20060101 A61K048/00; A61K 38/07 20060101
A61K038/07; A61K 38/31 20060101 A61K038/31; A61K 38/22 20060101
A61K038/22; A61K 38/28 20060101 A61K038/28; A61K 38/38 20060101
A61K038/38 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 20, 2008 |
CN |
PCT/CN2008/000551 |
Claims
1. A sustained-release pharmaceutical composition, comprising a
therapeutically effective amount of an active ingredient, an
amphipathic molecule, an organic acid and/or a salt thereof which
is hardly soluble in water, and an oily solvent.
2. The sustained-release pharmaceutical composition according to
claim 1, wherein the active ingredient is a hydrophilic drug.
3. The sustained-release pharmaceutical composition according to
claim 2, wherein the hydrophilic drug is selected from the group
consisting of peptides or proteins; nucleic acids; saccharides or
non-peptide non-nucleic acid organic drugs; and a mixture
thereof.
4. The sustained-release pharmaceutical composition according to
claim 3, wherein the peptides or proteins are selected from
pituitary polypeptides such as adrenal cortical hormone, gastrin,
vasopressin, oxytocin, melanoma stimulating hormone, and the like;
gastrointestinal peptides such as secretin, gastrin,
cholecystokinin, gastrone, vasoactive intestinal peptide,
pancreatic polypeptide, neurotensin, frog skin peptide, and the
like; hypothalamic peptides such as thyrotropin releasing hormone,
gonadotropin releasing hormone, somatostatin, growth hormone
releasing hormone, MSH cytokine inhibiting hormone, and the like;
brain peptides such as enkephalin, neoendorphine, endorphin, memory
peptide, and the like; kinins such as angiotensins I, II, III, and
the like; glutathione; calcitonin; sleep-inducing peptides; pineal
peptides; solcoseryl; thymosin; thymopentin; octreotide; exenatide;
pramlintide; fibrous proteins; fibrinogens; gastric mucin; gelatin;
gelatin sponge; protamines; endostatins; exendin; parotin; hirudin;
hepatocyte growth factors; leuprorelin; triptorelin; nafarelin;
goserelin; buserelin; bovine serum albumins; insulin;
erythropoietin (EPO); tumor necrosis factors; vaccines; auxins;
glucagons; serum albumins; gamma-globulins; trypsin inhibitors;
erythropoietins; interferons; interleukins; colony-stimulating
factors (GM-CSFs); luteinizing hormones, phytohemagglutinin,
trichosanthin, plant toxic proteins; and antibodies.
5. The sustained-release pharmaceutical composition according to
claim 3, wherein the nucleic acids include DNA fragments such as
DNA fragment comprising 33 base pair, chemically modified DNA
fragments such as thio-DNA fragments, RNA fragments, chemically
modified RNA fragments, polyinosinic acid, mecapto polycytidylic
acid, cAMP, CTP, CDP-choline, GMP, IMP, AMP, inosine, UTP, NAD,
NADP, 2-methylmercapto furan inosinic acid, bisformyl cAMP,
6-mercaptopurine, 6-mercaptopurinenucleoside, 6-thiopurine,
5-fluorouracil, furan fluorouracil, 2-deoxynucleoside, cytarabine
hydrochloride, and antiviral enzyme plasmid gene.
6. The sustained-release pharmaceutical composition according to
claim 3, wherein the saccharides or non-peptide non-nucleic acid
organic drugs are selected from polysaccharide drugs such as
heparin, pilose antler polysaccharides, polysaccharide from
stichopus japonicus, chitosan, dextran, lentinan, tremella
polysaccharide, pachymaran, ganoderma lucidum polysaccharides, and
the like; chemically synthesized drugs such as naltrexone
hydrochloride, morphine hydrochloride mitoxantrone hydrochloride,
cortisone acetate, and the like.
7. The sustained-release pharmaceutical composition according to
any one of claims 1-6, wherein the amount of the active ingredient
is from about 0.0001% to about 50% (weight percentage, w/w),
particularly from about 0.0005% to about 30% (w/w), particularly
from about 0.0005% to about 10% (w/w), particularly from about
0.0005% to about 5% (w/w), based on the total amount of the
composition.
8. The sustained-release pharmaceutical composition according to
claim 1, wherein the amphipathic molecule is a surfactant.
9. The sustained-release pharmaceutical composition according to
claim 8, wherein the surfactant is a non-ionic surfactant.
10. The sustained-release pharmaceutical composition according to
claim 9, wherein the non-ionic surfactant is selected from
polyethylene glycols such as fatty alcohol-polyoxyethylene ether
(AEO), alkylphenol ethoxylates, fatty acid ethoxylates,
polyoxyethylene fatty amine, ethylene xoide-propylene oxide block
copolymerized ethers, and the like; polyols such as monoalcohol
esters, ethylene glycol esters, glycerol esters, neopentyl-type
polyol esters, sorbitol esters, sorbitan esters, glycosyl esters,
alkyl glucosides, and the like; nitrogen-containing non-ionic
surfactants such as alkyl alcohol amides, amine oxides, and the
like; and sterol-derived non-ionic surfactants.
11. The sustained-release pharmaceutical composition according to
claim 8, wherein the surfactant is a phospholipid.
12. The sustained-release pharmaceutical composition according to
claim 11, wherein the phospholipid is selected from natural
phospholipids, including but not limited to phosphatidic acids,
phosphatidyl glycerol (PG), cardiolipin, phosphatidyl choline,
phosphatidyl ethanolamine, phosphatidyl serine (PS), phosphatidyl
inositol (PI), plasmalogens, ether lipids, phosphatidyl
ethanolamine (PE), soybean phosphatidyl choline (SPC) or eggyolk
phosphatidyl choline (EPC), phosphatidic acid (PA), sphingomyelin
(SPH), galactocerebroside, glucocerebroside, sulfatide,
ganglioside, and the like; synthetic phospholipids, including but
not limited to dipalmitoyl phosphatidyl choline (DPPC), distearoyl
phosphatidyl choline (DSPC), distearoyl phosphatidyl ethanolamine
(DSPE), hydrogenated soybean phosphatidyl choline (HSPC), PEGylated
distearoyl phosphatidyl ethanolamine (DSPE-PEG), and the like.
13. The sustained-release pharmaceutical composition according to
claim 12, wherein the phospholipid is selected from eggyolk
phosphatidyl choline (EPC) or hydrogenated soybean phosphatidyl
choline (HSPC).
14. The sustained-release pharmaceutical composition according to
claim 8, wherein the surfactant is a cholesterol.
15. The sustained-release pharmaceutical composition according to
claim 8, wherein the surfactant is any mixture of a non-ionic
surfactant, a phospholipid and a cholesterol.
16. The sustained-release pharmaceutical composition according to
claim 1, wherein the amount of the amphipathic molecule is from
about 0.0001% to about 30.0% (weight percentage, w/w), particularly
from about 0.005% to about 20% (w/w), particularly from about
0.005% to about 10% (w/w), based on the total amount of the
composition.
17. The sustained-release pharmaceutical composition according to
claim 1, wherein the organic acid and/or a salt thereof which is
hardly soluble in water is selected from lauric acid, myristic
acid, palmitic acid, oleic acid, linoleic acid, linolenic acid,
stearic acid, palmitic acid, arachidonic acid, pamoic acid and/or a
salt thereof.
18. The sustained-release pharmaceutical composition according to
claim 17, wherein the salt of the organic acid which is hardly
soluble in water is selected from a salt of calcium, magnesium,
barium, manganese, iron, copper, zinc, and aluminum of the organic
acid which is hardly soluble in water.
19. The sustained-release pharmaceutical composition according to
claim 1, wherein the amount of the organic acid and/or a salt
thereof which is hardly soluble in water is from about 0.0001% to
about 30% (weight percentage, w/w), from about 0.005% to about 20%
(w/w), from about 0.005% to about 10% (w/w), based on the total
amount of the composition.
20. The sustained-release pharmaceutical composition according to
claim 1, wherein the oily solvent is selected from the group
consisting of injectable natural plant oils, refined plant oils,
long-chain or medium-chain fatty acid glycerides, benzyl benzoate,
and a mixture thereof, and is preferably selected from injectable
soybean, or a long-chain or medium-chain fatty acid glycerides.
21. A sustained-release pharmaceutical formulation, comprising a
sustained-release pharmaceutical composition according to claim
1.
22. The sustained-release pharmaceutical formulation according to
claim 21, being an injectable sustained-release pharmaceutical
formulation.
23. A process for preparing a sustained-release pharmaceutical
formulation of claim 21, comprising: (1) dissolving or suspending
an active ingredient into an aqueous solvent; (2) dissolving or
suspending an amphipathic molecule and an organic acid and/or a
salt thereof which is hardly soluble in water into an organic
solvent; (3) dispersing the aqueous mixture of the active
ingredient obtained in step (1) into the organic mixture obtained
in step (2); (4) removing the organic solvent from the mixture
obtained in step (3); (5) drying the product obtained in step (4)
to form a solid; and (6) dissolving or suspending the solid
obtained in step (5) into an oily solvent.
24. The process according to claim 23, wherein an appropriate
amount of water is added to the solid formed after removing the
solvent in step (4) to disperse the solid to obtain a uniform
suspension.
25. The process according to claim 23, wherein the drying process
in step (5) is lyophilization.
26. The process according to claim 23, wherein the
sustained-release pharmaceutical formulation is an injectable
sustained-release pharmaceutical formulation.
27. A process for treating diseases in a subject, comprising
administrating to the subject a therapeutically effective amount of
a pharmaceutical composition of claim 1 or a sustained-release
pharmaceutical formulation of claim 21.
Description
CROSS REFERENCE OF RELATED APPLICATIONS
[0001] The present application claims the benefit of the
international application PCT/CN2008/000551, titled "Injectable
Sustained-Release Formulation and Process for Preparation thereof",
which was filed on Mar. 20, 2008, and of which all the contents are
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The present application relates to a sustained-release
pharmaceutical composition, in particular to a sustained-release
composition of hydrophilic biological drugs such as peptides,
proteins, nucleic acids, saccharides and the like. The present
application further relates to an injectable sustained-release
pharmaceutical formulation prepared from the sustained-release
pharmaceutical composition and to a process for preparing the
injectable sustained-release pharmaceutical formulation.
TECHNICAL BACKGROUND
[0003] With the fast development of biological technology,
biological drugs such as peptides, proteins, nucleic acids,
saccharides and the like are becoming a group of important
therapeutic agents.
[0004] Although the efficacy of biological drugs has been
demonstrated by clinical studies, comparing with small-molecule
drugs, biological drugs suffer from lower stabilities and are more
liable to deactivation. In addition, most of biological drugs
belong to hydrophilic large-molecule materials with low lipid/water
partition coefficient and are therefore difficult to be taken in by
lipophilic membranes, which results in that biological drugs are
difficult to pass biological barriers. Therefore, the oral
bio-availabilities of biological drugs are normally low.
[0005] Accordingly, for biological drugs, a good route of
administration is through parental administration, e.g. through
injection. However, for patients who need to maintain a certain
level of drug concentration in blood, this kind of administration
shall be performed repeatedly. Therefore, a sustained-release
formulation of biological drugs was developed recently in order to
improve the rationality and efficiency of the administration.
[0006] Suspensions or solutions prepared by dissolving drugs in
oily solvents would have sustained-release effects. However, when
drugs with high water-solubilities, e.g. biological drugs, are
suspended or partially dissolved in oils, the drugs are liable to
entering into the water phase when reaching the oil/water
interface. Therefore, for biological drugs which have high
water-solubilities or high polarities, sustained-release effects
are difficult to be achieved by simply utilizing oily
suspensions.
[0007] In certain therapeutic fields, liposome has been
successfully uses as the vehicle for releasing biological drugs.
However, as a sustained-release system; liposome still has some
issue to solve, for example, under certain conditions, the
sustained-release effect is not satisfactory, encapsulating ratio
is low, the physical and chemical stability is poor, etc.
[0008] Although a notable progress has been made to
sustained-release formulations of drugs such as peptides, proteins,
nucleic acids and saccharides, and some injectable
sustained-release formulations have been successfully marketed,
this kind of formulations in the art are still not satisfactory due
to their complex manufacturing process and rigid operational
requirements.
[0009] Accordingly, various new sustained-release pharmaceutical
formulations are still needed to meet different therapeutic
requirements.
SUMMARY
[0010] In one aspect, the present application relates to a
sustained-release pharmaceutical composition, comprising a
therapeutically effective amount of an active ingredient, an
amphipathic molecule, an organic acid and/or a salt thereof which
is hardly soluble in water, and an oily solvent.
[0011] In another aspect, the present application relates to an
injectable sustained-release pharmaceutical formulation prepared
from the sustained-release pharmaceutical composition disclosed
herein.
[0012] In a further aspect, the present application provides a
process for preparing a injectable sustained-release pharmaceutical
formulation, comprising:
[0013] (1) dissolving or suspending an active ingredient into an
aqueous solvent;
[0014] (2) dissolving or suspending an amphipathic molecule and an
organic acid and/or a salt thereof which is hardly soluble in water
into an organic solvent;
[0015] (3) dispersing the aqueous mixture of the active ingredient
obtained in step (1) into the organic mixture obtained in step
(2);
[0016] (4) removing the organic solvent from the mixture obtained
in step (3);
[0017] (5) drying the products obtained in step (4) to form a
solid; and
[0018] (6) dissolving or suspending the solid obtained in step (5)
into an oily solvent.
[0019] In a further aspect, the present application provides an
injectable sustained-release pharmaceutical formulation, which
comprises an active ingredient in a therapeutically effective
amount, an amphipathic molecule, an organic acid and/or a salt
thereof which is hardly soluble in water, and an oily solvent, the
injectable sustained-release pharmaceutical formulation is prepared
by the steps of:
[0020] (1) dissolving or suspending the active ingredient into an
aqueous solvent;
[0021] (2) dissolving or suspending the amphipathic molecule and
the organic acid and/or a salt thereof which is hardly soluble in
water into an organic solvent;
[0022] (3) dispersing the aqueous mixture of the active ingredient
obtained in step (1) into the organic mixture obtained in step
(2);
[0023] (4) removing the organic solvent from the mixture obtained
in step (3);
[0024] (5) drying the products obtained in step (4) to form a
solid; and
[0025] (6) dissolving or suspending the solid obtained in step (5)
into the oily solvent.
[0026] In a further aspect, the present application provides a
process for treating a subject, comprising administrating to the
subject a therapeutically effective amount of a pharmaceutical
composition or a sustained-release pharmaceutical formulation of
the present application.
[0027] The sustained-release pharmaceutical formulation of the
present application provides a good sustained-release effect for
hydrophilic biological drugs, in particular peptides, proteins,
nucleic acids and saccharides.
DETAILED DESCRIPTION OF EMBODIMENTS
[0028] In one aspect, the present application relates to a
sustained-release pharmaceutical composition, comprising an active
ingredient in a therapeutically effective amount, an amphipathic
molecule, and an organic acid and/or a salt thereof which is hardly
soluble in water
[0029] The active ingredient may be used in the composition of the
present application is a hydrophilic drug, including but not
limited to:
[0030] peptides and proteins, for example, pituitary polypeptides
such as adrenal cortical hormone, gastrin, vasopressin, oxytocin,
melanoma stimulating hormone, and the like; gastrointestinal
peptides such as secretin, gastrin, cholecystokinin, gastrone,
vasoactive intestinal peptide, pancreatic polypeptide, neurotensin,
frog skin peptide, and the like; hypothalamic peptides such as
thyrotropin releasing hormone, gonadotropin releasing hormone,
somatostatin, growth hormone releasing hormone, MSH cytokine
inhibiting hormone, and the like; brain peptides such as
enkephalin, neoendorphine, endorphin, memory peptide, and the like;
kinins such as angiotensins I, II, III, and the like; glutathione;
calcitonin; sleep-inducing peptides; pineal peptides; solcoseryl;
thymosin; thymopentin; octreotide; exenatide; pramlintide; fibrous
proteins; fibrinogens; gastric mucin; gelatin; gelatin sponge;
protamines; endostatins; exendin; parotin; hirudin; hepatocyte
growth factors; leuprorelin; triptorelin; nafarelin; goserelin;
buserelin; bovine serum albumins; insulin; erythropoietin (EPO);
tumor necrosis factors; vaccines; auxins; glucagons; serum
albumins; gamma-globulins; trypsin inhibitors; erythropoietins;
interferons; interleukins; colony-stimulating factors (GM-CSFs);
luteinizing hormones, phytohemagglutinin, trichosanthin, plant
toxic proteins; antibodies, and the like;
[0031] nucleic acids, for example, DNA fragments such as DNA
fragment comprising 33 base pair, chemically modified DNA fragments
such as thio-DNA fragments, RNA fragments, chemically modified RNA
fragments, polyinosinic acid, mecapto polycytidylic acid, cAMP,
CTP, CDP-choline, GMP, IMP, AMP, inosine. UTP, NAD, NADP,
2-methylmercapto furan inosinic acid, bisformyl cAMP,
6-mercaptopurine, 6-mercaptopurinenucleoside, 6-thiopurine,
5-fluorouracil, furan fluorouracil, from organic bases include but
not limited to salts of isopropylamine, diethylamine,
1,2-diaminoethane, ethanolamine, diethanolamine, trimethylamine,
dicylcohexylamine, choline, caffeine, and the like.
[0032] In some preferred embodiments, the active ingredient in the
composition of the present application may be leuprorelin acetate,
or triptorelin acetate.
[0033] Other pharmaceutically acceptable derivatives of the active
ingredient are those well-known to a person skilled in the art,
including but not limited to prodrugs thereof.
[0034] "Prodrug" refers to a compound which can be converted to an
active ingredient through solvent decomposition under physiological
conditions. Accordingly, the term "prodrug" refers to a
pharmaceutically acceptable metabolic precursor of the active
ingredient in the composition of the present application. Examples
of the prodrug include but not limited, to acetate, formate,
benzoate, phosphate, sulfonates derivatives of the alcohol
functionality; and ester or amide derivatives of the carboxylic
acid functionality, of the active ingredient in the composition of
the present application.
[0035] The amount of the active ingredient comprised in the
composition of the present application is based on achieving a
therapeutically effective amount.
[0036] "Therapeutically effective amount" refers to the amount of
the active ingredient in the composition of the present
application, which is sufficient to achieve treatment/prevention of
a disease or condition to be treated/prevented in a mammal,
especially human being, when it is administered thereto. The amount
of the active ingredient in the composition of the present
application constituting a "therapeutically effective amount" may
vary according to the type of the active ingredient, the condition
and the severity thereof, and the physical conditions of the
subject such as age, weight and the like, and may conventionally
determined by a person with ordinary skill in the art according to
their own knowledge and the disclosure of the present
application.
[0037] The active ingredient may be a single drug, or a combination
of one or more pharmaceutically compatible drugs.
[0038] The amount of the active ingredient in the composition of
the present application is normally from about 0.0001% to about 50%
based on the total amount of the composition (weight percentage,
w/w). In some embodiments, the amount of the active ingredient in
the 2-deoxynucleoside, cytarabine hydrochloride, antiviral enzyme
plasmid gene, and the like;
[0039] saccharides, and non-peptide non-nucleic acid organic drugs,
for example, polysaccharide drugs such as heparin, pilose antler
polysaccharides, polysaccharide from stichopus japonicus, chitosan,
dextran, lentinan, tremella polysaccharide, pachymaran. ganoderma
lucidum polysaccharides, and the like; chemically synthesized drugs
such as naltrexone hydrochloride, morphine hydrochloride,
mitoxantrone hydrochloride, cortisone acetate, and the like.
[0040] In some preferred embodiment's, the active ingredient in the
composition of the present application may include peptides and
proteins. In some more preferred embodiments, the active ingredient
in the composition of the present application may be selected from
the group consisting of thymopentin, bovine serum albumins,
exenatide, pramlintide, somatostatin, .omega.-interferons,
octreotide, salmon calcitonin, and insulin.
[0041] In some preferred embodiments, the active ingredient in the
composition of the present application may be nucleic acids. In
some more preferred embodiments, the active ingredient in the
composition of the present application may be selected from
oligonucleotide.
[0042] In some preferred embodiments, the active ingredient in the
composition of the present application may be saccharides and
non-peptide non-nucleic acid organic drugs. In some more preferred
embodiments, the active ingredient in the composition of the
present application may be selected from naltrexone
hydrochloride.
[0043] In some embodiments of the present application, the active
ingredient may be pharmaceutically acceptable salts or other
derivatives thereof.
[0044] The pharmaceutically acceptable salts of the active
ingredient are those well-known to a person skilled in the art,
including acid addition salts and base addition salts. Exemplary
acids include inorganic salts such as hydrochloric acid, sulfuric
acid, phosphoric acid, hydrobromic acid, boric acid, and the like;
and organic acids such as acetic acid, maleic acid, tartaric acid,
salicylic acid, citric acid, benzoic acid, pamoic acid, sulfonic
acid, and the like. Exemplary bases include inorganic bases and
organic bases. Salts derived from inorganic bases are those
well-known to a person skilled in the art, including but not
limited to ammonium, sodium, potassium, calcium, magnesium, and the
like. Salts derived composition of the present application is from
about 0.0005% to about 30% based on the total amount of the
composition (w/w). In some embodiments, the amount of the active
ingredient in the composition of the present application is from
about 0.0005% to about 10% based on the total amount of the
composition (w/w). In some embodiments, the amount of the active
ingredient in the composition of the present application is from
about 0.0005% to about 5% based on the total amount of the
composition (w/w).
[0045] The amphipathic molecule of the present application may be
any molecule having both a hydrophilic group and a hydrophobic
group. The amphipathic molecule includes surfactants and other
materials which have surface activity, such as short chain fatty
acids or fatty alcohols.
[0046] In some preferred embodiments, the amphipathic molecule used
in the present application may be a surfactant.
[0047] The surfactant used in the present application may be an
ionic surfactant or a non-ionic surfactant conventionally used in
the pharmaceutics.
[0048] The ionic surfactant includes anionic surfactants, cationic
surfactants and amphipathic surfactants.
[0049] In some embodiments of the present application, for ionic
surfactants, those having low water-solubility are preferred.
[0050] Exemplary ionic surfactants include but not limited to
anionic surfactants such as salts of fatty acids, sulfated
compounds, sulfonated compounds, and the like; cationic surfactants
such as quaternary ammonium compounds, and the like; and
amphipathic surfactants such as amino acids, betaines, and the
like.
[0051] Exemplary non-ionic surfactants include but not limited to
polyethylene glycols such as fatty alcohol-polyoxyethylene ether
(AEO), alkylphenol ethoxylates, fatty acid ethoxylates,
polyoxyethylene fatty amine, ethylene xoide-propylene oxide block
copolymerized ethers, and the like; polyols such as monoalcohol
esters, ethylene glycol esters, glycerol esters, neopentyl-type
polyol esters, sorbitol esters, sorbitan esters, glycosyl esters,
alkyl glucosides, and the like; nitrogen-containing non-ionic
surfactants such as alkyl alcohol amides, amine oxides, and the
like; and sterol-derived non-ionic surfactants.
[0052] In some embodiments, the surfactant used in the present
application may be a phospholipid. The phospholipid used in the
present application is selected from natural phospholipids,
including but not limited to phosphatidic acids, phosphatidyl
glycerol (PG), cardiolipin, phosphatidyl choline, phosphatidyl
ethanolamine, phosphatidyl serine (PS), phosphatidyl inositol (PI),
plasmalogens, ether lipids, phosphatidyl ethanolamine (PE), soybean
phosphatidyl choline (SPC) or eggyolk phosphatidyl choline (EPC),
phosphatidic acid (PA), sphingomyelin (SPH), galactocerebroside,
glucocerebroside, sulfatide, ganglioside, and the like; synthetic
phospholipids, including but not limited to dipalmitoyl
phosphatidyl choline (DPPC), distearoyl phosphatidyl choline
(DSPC), distearoyl phosphatidyl ethanolamine (DSPE), hydrogenated
soybean phosphatidyl choline (HSPC), PEGylated distearoyl
phosphatidyl ethanolamine (DSPE-PEG), and the like. In some
preferred embodiments, the phospholipids is eggyolk phosphatidyl
choline (EPC) or hydrogenated soybean phosphatidyl choline
(HSPC).
[0053] In some embodiments, the surfactant used in the present
application may be cholesterols. In some preferred embodiments, the
surfactant used in the present application may be cholesterol.
[0054] The amphipathic molecule added into the composition of the
present application may be a mixture formed by combining one or
more of the above surfactants.
[0055] In some embodiments, the surfactant used in the present
application may also be a mixture of eggyolk phosphatidyl choline
(EPC) and cholesterol.
[0056] The selection of a specific amphipathic molecule in the
composition depends on various factors, such as the type, polarity
and pH of the active molecule, the type and concentration of other
additives existing in the composition, and the like. However, a
person skilled in the art is able to perform the selection
according to specific conditions of the composition. The selection
and amount of the specific amphipathic molecule are based on
forming a lipid-drug complex particulate.
[0057] The amount of the specific amphipathic molecule is normally
from about 0.0001% to about 30.0% based on the total amount of the
composition (weight percentage, w/w). In some embodiments, the
amount of the specific amphipathic molecule is front about 0.005%
to about 20% based on the total amount of the composition (w/w). In
some embodiments, the amount of the specific amphipathic molecule
is from about 0.005% to about 10% based on the total amount of the
composition (w/w).
[0058] In addition to the amphipathic molecule, an organic acid
and/or a salt thereof which is hardly soluble in water is also
added into the sustained-release pharmaceutical composition of the
present application. Therefore, the sustained-release performance
is significantly improved. Although not verified by any theory, it
is presumed that, in one hand, the active ingredient interacts with
the organic acid and/or a salt thereof which is hardly soluble in
water through electrostatic force, hydrophobic interaction, and
coordination bonding to improve the lipophilicity and stability of
the active ingredient and delay the release of the drug; in the
other hand, adding an organic acid and/or a salt thereof which is
hardly soluble in water into the composition would facilitate the
dispersing of the formed lipid-drug complex in the oily
solvent.
[0059] In some embodiments, the organic acid and/or a salt thereof
which is hardly soluble in water is preferably that which is in the
form of a solid under pharmaceutical conditions. In some
embodiments, salts of organic acids are preferred.
[0060] The term "hardly soluble in water" as used herein refers to
that the solubility of the organic acid or the salt thereof in 100
g water is less than or equal to 1 g.
[0061] In some embodiments, the organic acid and/or a salt thereof
which is hardly soluble in water used in the composition of the
present application may be selected from aliphatic acids or
aromatic acids.
[0062] Exemplary organic acids include by not limited to saturated
or unsaturated aliphatic acids having more than 10 carbon atoms,
such as lauric acid, myristic acid, palmitic acid, oleic acid,
linoleic acid, linolenic acid, stearic acid, palmitic acid,
arachidonic acid, and the like. Exemplary aromatic acids include
pamoic acid.
[0063] A salt of the organic acid which is hardly soluble in water
may be selected from any salt of an organic acid which is hardly
soluble in water, including but not limited to a salt of calcium,
magnesium, barium, manganese, iron, copper, zinc, and aluminum, or
may be a salt formed from any other organic acid, provided that it
is hardly soluble in water and must be pharmaceutically acceptable
(non-toxic).
[0064] In the composition of the present application, the organic
acid which is hardly soluble in water, the salt of the organic acid
which is hardly soluble in water, or a mixture thereof may be
used.
[0065] In some embodiments, the organic acid and/or a salt thereof
which is hardly soluble in water may be a combination of one or
more of the above.
[0066] The specific amount of the organic acid and/or a salt
thereof which is hardly soluble in water is normally from about
0.0001% to about 30% based on the total amount of the composition
(weight percentage, w/w). In some embodiments, the specific amount
of the organic acid and/or a salt thereof which is hardly soluble
in water is from about 0.005% to about 20% based on the total
amount of the composition (w/w). In some embodiments, the specific
amount of the organic acid and/or a salt thereof which is hardly
soluble in water is from about 0.005% to about 10% based on the
total amount of the composition (w/w).
[0067] The sustained-release pharmaceutical composition of the
present application may further comprise a pharmaceutically
acceptable vehicle or excipient. Preferably, the vehicle or
excipient is an oily solvent.
[0068] The oily solvent of the composition of the present
application may be that conventionally used in the pharmaceutical
field which is well-known to the person skilled in the art.
Exemplary oily solvents include but not limited to natural plant
oils such as soybean oil, Camellia oil, sesame oil, garlic oil,
walnut oil, olive oil, corn oil, peanut oil, coconut oil,
cottonseed oil, castor oil, and the like; refined plant oils;
long-chain or medium-chain fatty acid glyceride; isopropyl
myristate; ethyl linoleate; polyoxyethylene triolein; white oil;
benzyl benzoate, and the like.
[0069] In some embodiments, the oily solvent may be a combination
of one or more of the above.
[0070] In some preferred embodiments, the oily solvent may be
soybean oil, or long-chain or medium-chain fatty acid
glyceride.
[0071] The amount of the oily solvent is not strict, which may be
selected by a person skilled in the art according to specific
dosage form. The amount of the oily solvent is normally about 5% to
about 99% of the total weight of the composition (weight
percentage, w/w). In some embodiments, the amount of the oily
solvent is about 30% to about 99% of the total weight of the
composition (weight percentage, w/w). In some embodiments, the
amount of the oily solvent is about 60% to about 99% of the total
weight of the composition (weight percentage, w/w).
[0072] In some embodiments, the sustained-release formulation of
the present application may further comprise a thickener. The
thickener which may be used in the present application includes
polymers such as PCL, PLGA, PLA, and the like. The amount of the
thickener is from about 0.05% to about 10%, preferably about 0.5%
to about 3.0%, based on the total weight of the sustained-release
formulation (w/w).
[0073] In some embodiments, the sustained-release formulation of
the present application may further comprise an antioxidant to
ensure the stability of the injectable oil. The antioxidant which
may be used in the present application may be selected from the
group consisting of VE (vitamin E), BHT (butylated hydroxy toluene)
BHA (butyl hydroxy anisd) and a mixture thereof. The amount of the
antioxidant is from about 0.01% to about 2.0% (w/w), preferably
about 0.05% to about 1.0%, based on the total weight of the
sustained-release formulation (w/w).
[0074] A person skilled in the art would appreciate that the type
and amount of the active ingredient, the amphipathic molecule, the
organic acid and/or a salt thereof which is hardly soluble in
water, and the oily solvent of the composition of the present
application may be optionally combined according to the above
ranges, provided that such a combination is able to achieve the
object of the present application.
[0075] In some embodiments of the present application, the
sustained-release pharmaceutical composition comprises from about 1
.mu.g to about 500 mg of a peptide or protein thug, from about 1
.mu.g to about 300 mg of a surfactant, from about 1 .mu.g to about
300 in of a saturated or unsaturated aliphatic acid having more
than 10 carbon atoms, and about 1 g of a natural plant oil.
[0076] In some embodiments of the present application, the
sustained-release pharmaceutical composition comprises from about 5
.mu.g to about 300 mg of a pharmaceutically acceptable salt of a
peptide or protein drug, from about 50 .mu.g to about 200 mg of a
surfactant, from about 50 .mu.g to about 200 mg of a salt which is
hardly soluble in water of a saturated or unsaturated aliphatic
acid having more than 10 carbon atoms, and about 1 g of a
long-chain or medium-chain fatty acid glyceride.
[0077] In some embodiments of the present application, the
sustained-release pharmaceutical composition comprises from about 5
.mu.g to about 100 mg of a peptide or protein drug, from about 50
.mu.g to about 100 mg of phospholipids-type surfactant, from about
50 .mu.g to about 100 mg of a saturated or unsaturated aliphatic
acid having more than 10 carbon atoms, and about 1 g of a
long-chain or medium-chain fatty acid glyceride.
[0078] In some embodiments of the present application, the
sustained-release pharmaceutical composition comprises from about 5
.mu.g to about 50 mg of a nucleic acid drug, from about 1 .mu.g to
about 300 mg of a phospholipids-type surfactant, from about 1 .mu.g
to about 300 mg of an aromatic acid which is hardly soluble in
water, and about 1 g of a long-chain or medium-chain fatty acid
glyceride.
[0079] In some embodiments of the present application, the
sustained-release pharmaceutical composition comprises from about 1
.mu.g to about 500 mg of a saccharide or a non-peptide non-nucleic
acid organic drug, from about 50 .mu.g to about 200 mg of a
phospholipids-type surfactant, from about 50 .mu.g to about 200 mg
of a salt of a saturated or unsaturated aliphatic acids having more
than 10 carbon atoms, and about 1 g of a natural plant oil.
[0080] In some embodiments of the present application, the
sustained-release pharmaceutical composition comprises from about 1
.mu.g to about 500 mg of salmon calcitonin, from about 1 .mu.g to
about 200 mg of a natural phospholipids, from about 1 .mu.g to
about 50 mg of cholesterol, from about 1 .mu.g to about 300 mg of a
salt which is hardly soluble in water of a saturated or unsaturated
aliphatic acid having more than 10 carbon atoms, and about 1 g of
an oily solvent.
[0081] In some embodiments of the present application, the
sustained-release pharmaceutical composition comprises from about 1
.mu.g to about 500 mg of exenatide, from about 1 .mu.g to about 200
mg of a natural phospholipids, from about 1 .mu.g to about 50 mg of
cholesterol, from about 1 .mu.g to about 300 mg of a salt which is
hardly soluble in water of a saturated or unsaturated aliphatic
acid having more than 10 carbon atoms, and about 1 g of an oily
solvent.
[0082] In some embodiments of the present application, the
sustained-release pharmaceutical composition comprises from about 1
.mu.g to about 500 mg of insulin, from about 1 .mu.g to about 200
mg of a natural phospholipids, from about 1 .mu.g to about 50 mg of
cholesterol, from about 1 .mu.g to about 300 mg of a salt which is
hardly soluble in water of a saturated or unsaturated aliphatic
acid having more than 10 carbon atoms, and about 1 g of an oily
solvent.
[0083] In another aspect, the present application relates to an
injectable sustained-release pharmaceutical formulation prepared
from a sustained-release pharmaceutical composition disclosed
above. The sustained-release pharmaceutical formulation may be
administered through any route which is considered appropriate by a
person skilled in the art. Preferably, the sustained-release
pharmaceutical formulation is an injectable sustained-release
pharmaceutical formulation. A person skilled in the art would
appreciate that when the sustained-release pharmaceutical
formulation is administered through injection, the components in
the formulation shall be injectable components.
[0084] In a further aspect, the present application provides a
process for preparing a sustained-release pharmaceutical
formulation, comprising:
[0085] (1) dissolving or suspending an active ingredient into an
aqueous solvent;
[0086] (2) dissolving or suspending an amphipathic molecule and an
organic acid and/or a salt thereof which is hardly soluble in water
into an organic solvent;
[0087] (3) dispersing the aqueous mixture of the active ingredient
obtained in step (1) into the organic mixture obtained in step
(2);
[0088] (4) removing the organic solvent from the mixture obtained
in step (3);
[0089] (5) drying the products obtained in step (4) to form a
solid; and
[0090] (6) dissolving or suspending the solid obtained in step (5)
into an oily solvent.
[0091] A person skilled in the art would appreciate that steps (1)
and (2) in the above process may not necessarily performed in the
indicated sequence.
[0092] The aqueous solvent used in step (1) includes but is not
limited to water, 0.9% sodium chloride aqueous solution, and any
pharmaceutically suitable aqueous buffer. In some preferred
embodiments, injectable water is used as an aqueous solvent. In
some preferred embodiments. PBB buffer is used as an aqueous
solvent.
[0093] The organic solvent used in step (2) may be selected from
any organic solvent which has good solubilizing effects on the
amphipathic molecule and the organic acid and/or salt thereof which
is hardly soluble in water, and has low boiling point to enable it
to be removed easily. Examples of the above organic solvents
include but are not limited to dichloromethane, chloroform, ethyl
ether, ethanol, methanol, n-propanol, iso-propanol, n-butanol,
tert-butanol, acetone, acetonitrile, ethyl acetate. Different
solvents may be selected according to the structure of the
amphipathic molecule and the organic acid and/or a salt thereof
which is hardly soluble in water used. The selection of the solvent
is well-known to a person skilled in the art. In some preferred
embodiments, dichloromethane is used as an organic solvent.
[0094] In step (3), in the preparation of a lipid-drug complex
particulate, an active drug may be completely encapsulated in the
lipid-drug complex particulate through processes such as ultrasonic
dispersion method, reverse evaporation technique, film dispersion
method, injection method, MVL preparation method, pH-gradient
method, ammonium sulfate gradient method, or second encapsulation
method, according to the nature of the active ingredient. In this
step, it is important to uniformly mix and disperse the aqueous
solution and the organic solution. In some preferred embodiments,
ultrasonic dispersion method is employed.
[0095] In step (3), the operation temperature is selected according
to the type of the amphipathic molecule used, the boiling point of
the organic solvent used. Normally, the preparation process is
carried out under a temperature in the range from -40.degree. C. to
45.degree. C. In some embodiments where HSPC is used as the
amphipathic molecule, the process may be carried out under a
temperature in the range from 40.degree. C. to 45.degree. C.
[0096] In step (4), the organic solvent is removed preferably
through evaporation under reduced pressure to prevent degradation
of the active ingredient in the formulation.
[0097] In some preferred embodiments, an appropriate amount of
water may be added to the solid formed after removing the solvent
to disperse the solid to obtain a uniform suspension, before the
drying in step (5) is performed.
[0098] The drying process in step (5) may be lyophilization, spray
drying or any other suitable drying process. The composition after
drying is in the form of a solid.
[0099] In the lyophilization, a cryoprotectant is normally used to
reduce the damage to the lipid-drug complex particulate during the
freezing and melting process and to the leaking of the drug during
the lyophilization. The effect of the cryoprotectant is to reduce
the breaking of the bi-molecule layer membrane during the
lyophilization, and to enable the lyophilized lipid particulate
encapsulating the drug to be readily dispersed in the oily media.
However, in the technical solution of the present application, the
salt of the organic acid which is hardly soluble in water may play
a role of cryoprotectant in addition of the role as disclosed
above. Therefore, in some embodiments of the present application,
there is no need to add any further cryoprotectant.
[0100] The solid obtain in the above step (5) is dissolved or
dispersed in an oily solvent to form a solution or a
suspension.
[0101] In the preparation process above, the sustained-release
pharmaceutical formulation is preferably an injectable
sustained-release formulation.
[0102] The present application may be used for biological drugs, or
may be used for any hydrophilic injectable drugs such as small
molecule compounds. The present application is particularly
suitable for drugs such as peptides, proteins, nucleic acids and
saccharides which have high polarity, good water-solubility and are
unstable in water. Sustained-release formulations of various drugs
such as peptides, proteins, nucleic acids were prepared herein with
this technique and these formulations show sustained-release effect
of 3 to 7 days in vitro. This type of the sustained-release
pharmaceutical formulation may be preferably administered through
intramuscular injection or subcutaneous injection, and keep
releasing the active ingredient for 3 to 7 days.
[0103] The present application is further illustrated with the
examples below. It shall be appreciated that these examples do not
constitute any limitation to the scope of the present
application.
EXAMPLES
Materials and Reagents
Active Ingredients
[0104] Leuprorelin acetate: synthesized in the inventor's
laboratory following a previously disclosed process (J. A.
Vilchez-Martinez, et al. Biochem. Biophys. Res. Commun. 1974.
59:1226), HPLC purity >98%;
[0105] Naltrexone hydrochloride: presented by Wellso Parmaceutical
Co. Ltd. China;
[0106] Thymopentin: synthesized in the inventor's laboratory
following a previously disclosed process (G. Goldstein, et al.
Science 1979, 204:1309). HPLC purity >98%;
[0107] Bovine serum albumins: purchased from Sigma, USA;
[0108] D33: DNA fragment containing 33 base pairs; 5'-d(TGC TCT CCA
GGC TAG CTA CAA CGA CCT GCA CCT)-3', synthesized in the inventor's
laboratory following a previously disclosed process (Naruhisa Ota,
et al. Nucleic Acid Research, 1998, 26(4):3385), HPLC purity
>98%; all the base pairs used in the synthesis of D33 were
purchased from Proligo LLC;
[0109] Exenatide: synthesized in the inventor's laboratory
following a previously disclosed process (U.S. Pat. No. 6,528,486),
HPLC purity >98%;
[0110] Pramlintide: synthesized in the inventor's laboratory
following a previously disclosed process (U.S. Pat. No. 5,998,367),
HPLC purity >98%;
[0111] Triptorelin acetate: synthesized in the inventor's
laboratory following a previously disclosed process (D. H. Coy, et
al. J Med. Chem. 1976, 19:423), HPLC purity >98%;
[0112] Somatostatin: synthesized in the inventor's laboratory
following a previously disclosed process (A. M. Felix, et al. Int.
J. Peptide Protein Res. 1980, 15:342), HPLC purity >98%;
[0113] .omega.-Interferon: presented by Southwest Pharmaceutical
Co., Ltd., China;
[0114] Octreotide: synthesized in the inventor's laboratory
following a previously disclosed process (W. Bauer, et al. Life
Sci. 1982, 31:1133), HPLC purity >98%;
[0115] Salmon calcitonin: synthesized in the inventors laboratory
following a previously disclosed process (U.S. Pat. No. 3,926,938),
HPLC purity >98%;
[0116] Insulin: purchased from Tonghua Dongbao Pharmaceutical Co.
Ltd., China;
Amphiphilic Molecules
[0117] Eggyolk phosphatidyl choline (EPC), hydrogenated soybean
phosphatidyl choline (HSPC), cholesterol: all purchased from
Shanghai Toshisun Enterprise Co. Ltd. China;
[0118] Span 85: purchased from Fisher, USA;
Aliphatic Acids and Salts Thereof
[0119] Aluminum stearate: purchased from Shanghai Bangcheng
Chemical Co. Ltd., China;
[0120] Stearic acid: purchased from Beijing Shunyi Lisui Chemical
Plant, China;
[0121] Oleic acid: purchased from Beijing Jinlong Chemical Reagents
Co. Ltd., China;
[0122] Zinc stearate: purchased from Tianjin Langhu Chemical
Engineering Co. Ltd., China;
Oily Solvents
[0123] Injectable medium-chain oil, injectable soybean oil, both
purchased from Tieling Beiya Pharmaceutical Oil Co. Ltd.,
China;
Other Reagents
[0124] Ethyl ether: purchased from Tianjin Third Chemical Reagents
Factory, China;
[0125] Methanol, dichloromethane: purchased from Beijing Chemical
Plant, China;
[0126] PBS buffer: formulated following the appendix of Chinese
Pharmacopoeia 2005;
[0127] Injectable water: purchased from Beijing Yahua
Pharmaceutical Co. Ltd., China.
Determination of In Vitro Accumulated Release
[0128] An active ingredient control was added into water to prepare
standard active ingredient solutions with concentrations of 10
.mu.g/mL, 20 .mu.g/mL, 30 .mu.g/mL, 50 .mu.g/mL, 100 .mu.g/mL, and
200 .mu.g/mL. Absorbance A was determined for each solution with
Folin-Ciocalteu method. The absorbance A was normalized with
concentration to establish a standard curve regression
equation.
[0129] An appropriate amount of the prepared sustained-release
pharmaceutical formulation was placed into a 50 mL conical beaker
equipped with a stopper, into which 10 mL of pH 7.10 phosphate
buffer was added. The conical beaker was shaken in a rocking bed
under a constant temperature of 37.+-.1.degree. C. and the shaking
frequency was 70 r/m. At different time point, fixed amount of 200
.mu.L of the sample was taken, and 200 .mu.L of pH 7.10 phosphate
buffer was added to make up the volume. The sample was centrifuged
under 12,000 r/m for 10 mins. The supernatant fluid was taken as a
sample solution. The absorbance of the sample solution was
determined with the same method as described above. The
concentration of the active ingredient was calculated with the
regression equation obtained above.
[0130] The calculated accumulated amount of the drug was compared
with the total amount of the added drug to calculate the percentage
of accumulated release of the drug.
Example 1
Preparation and Sustained-Release Effects of Injectable
Sustained-Release Formulation of Leuprorelin Acetate
[0131] 1 mg of leuprorelin acetate was dissolved in 5 mL of 10
mmol/L PBS buffer (pH 7.0) as an aqueous phase. 20 mg of eggyolk
phosphatidyl choline (EPC), 5 mg of cholesterol and 20 mg of
aluminium stearate were dissolved in 20 mL of ethyl ether-methanol
(10:1) mixed solvent as an organic phase. The above aqueous phase
was dropwise added into the above organic phase at 30.degree. C.
under sufficient stirring. The resultant mixture was then treated
in a water bath ultrasonic unit until a uniform emulsion system was
formed. The mixture was evaporated under reduced pressure to remove
the organic solvents and an appropriate amount of water was added
to uniformly disperse the solid. The obtained suspension was
lyophilized to remove water. 1 g of injectable medium-chain oil was
added into the obtained solid product and stirred to disperse
uniformly.
[0132] Following the above method for determining the in vitro
accumulated release, the results of the in vitro accumulated
release of the prepared sustained-release formulation of
leuprorelin acetate in 7 days were determined and listed below:
TABLE-US-00001 1 day 3 days 5 days 7 days 20.6% 37.4% 76.0%
94.0%
Example 2
Preparation and Sustained-Release Effects of Injectable
Sustained-Release Formulation of Naltrexone Hydrochloride
[0133] 2 mg of naltrexone hydrochloride was dissolved in 5 mL of
injectable water as an aqueous phase. 20 mg of hydrogenated soybean
phosphatidyl choline (HSPC). 5 mg of cholesterol and 20 mg of
aluminium stearate were dissolved in 20 mL of dichloromethane as an
organic phase. The above aqueous phase was dropwise added into the
above organic phase at 44.degree. C. under sufficient stirring. The
resultant mixture was then treated in a water bath ultrasonic unit
until a uniform emulsion system was formed. The mixture was
evaporated under the reduced pressure to remove the organic solvent
and the obtained suspension was lyophilized to remove water. 1 g of
injectable medium-chain oil was added into the obtained solid
product and stirred to disperse uniformly.
[0134] Following the above method for determining the in vitro
accumulated release, the results of the in vitro accumulated
release of the prepared sustained-release formulation of naltrexone
hydrochloride in 7 days were determined and listed below:
TABLE-US-00002 1 day 3 days 5 days 7 days 35.9% 56.4% 78.2%
96.3%
Example 3
Preparation and Sustained-Release Effects of Injectable
Sustained-Release Formulation of Oligonucleotide
[0135] 2 mg of D33 was dissolved in 5 mL of injectable water as an
aqueous phase. 20 mg of EPC, 5 mg of cholesterol and 20 mg of
aluminium stearate were dissolved in 20 mL of dichloromethane as an
organic phase. The above aqueous phase was dropwise added into the
above organic phase at 30.degree. C. under sufficient stirring. The
resultant mixture was then treated in a water bath ultrasonic unit
until a uniform emulsion system was formed. The mixture was
evaporated under the reduced pressure to remove the organic solvent
and the obtained suspension was lyophilized to remove water. 1 g of
injectable medium-chain oil was added into the obtained solid
product and stirred to disperse uniformly.
[0136] Following the above method for determining the in vitro
accumulated release, the results of the in vitro accumulated
release of the prepared sustained-release formulation of
oligonucleotide in 7 days were determined and listed below:
TABLE-US-00003 1 day 3 days 5 days 7 days 38.8% 48.2% 54.1%
64.7%
Example 4
[0137] Following the same procedure as in Example 1, using
different amphipathic molecules, different organic acids or salts
which are hardly soluble in water, and different conditions of
preparation, sustained-release formulations of different active
ingredients were prepared, as shown in Table 1. The results of the
in vitro accumulated release in 7 days were determined and listed
in Table 2.
TABLE-US-00004 TABLE 1 Sustained-release formulations of different
active ingredients Organic acids or salts which are Aqueous Active
Amphipathic hardly soluble in Organic solvents Aqueous phase
phase/organic Oily solvents ingredients and molecules and water and
amounts and amounts solvent and phase mixing and amounts Nos.
amounts thereof amounts thereof thereof thereof amounts thereof
temperature (.degree. C.) thereof 1 Leuprorelin EPC 20 mg Stearic
acid Ethyl ether Injectable water 20 Injectable acetate 2 mg
Cholesterol 5 mg 5 mg 20 mL 5 mL soybean oil 1 g 2 Leuprorelin EPC
20 mg Oleic acid Dichloromethane 10 mmol/L pH 30 Injectable acetate
2 mg Cholesterol 5 mg 5 mg 20 mL 7.0 PBS buffer soybean 5 mL oil 1
g 3 Thymopentin HSPC 20 mg Zinc stearate Dichloromethane 10 mmol/L
pH 44 Injectable 1 mg Cholesterol 5 mg 20 mg 20 mL 7.0 PBS buffer
medium-chain 5 mL oil 1 g 4 Bovine serum EPC 20 mg Aluminium
stearate Dichloromethane Injectable water 30 Injectable albumin 1
mg Cholesterol 5 mg 25 mg 20 mL 5 mL medium-chain oil 1 g 5
Leuprorelin Span 85 20 mg Aluminium stearate Ethyl ether 10 mmol/L
pH 20 Injectable acetate 1 mg Cholesterol 5 mg 25 mg 20 mL 7.0 PBS
buffer soybean 5 mL oil 1 g 6 Exenatide EPC 20 mg Zinc stearate
Dichloromethane Injectable water 30 Injectable 2 mg Cholesterol 5
mg 20 mg 20 mL 5 mL medium-chain oil 1 g 7 Pramlintide EPC 20 mg
Aluminium stearate Dichloromethane Injectable water 30 Injectable 2
mg Cholesterol 5 mg 20 mg 20 mL 5 mL medium-chain oil 1 g 8
Triptorelin EPC 20 mg Aluminium stearate Dichloromethane Injectable
water 30 Injectable acetate 2 mg Cholesterol 5 mg 20 mg 20 mL 5 mL
medium-chain oil 1 g 9 Somatostatin EPC 20 mg Aluminium stearate
Dichloromethane Injectable water 30 Injectable 2 mg Cholesterol 5
mg 20 mg 20 mL 5 mL medium-chain oil 1 g 10 .omega.-Interferons EPC
20 mg Aluminium stearate Dichloromethane Injectable water 30
Injectable 2 mg Cholesterol 5 mg 20 mg 20 mL 5 mL medium-chain oil
1 g 11 Octreotide EPC 20 mg Aluminium stearate Dichloromethane
Injectable water 30 Injectable 5 mg Cholesterol 5 mg 20 mg 20 mL 5
mL medium-chain oil 1 g 12 Salmon EPC 20 mg Aluminium stearate
Dichloromethane Injectable water 30 Injectable calcitonin 1 mg
Cholesterol 5 mg 20 mg 20 mL 5 mL medium-chain oil 1 g 13 Insulin
EPC 20 mg Aluminium stearate Dichloromethane Injectable water 30
Injectable 1 mg Cholesterol 5 mg 20 mg 20 mL 5 mL medium-chain oil
1 g
TABLE-US-00005 TABLE 2 In vitro release results of different
sustained-release pharmaceutical formulations Accumulated release
of drugs % Nos. 1 day 3 days 5 days 7 days 1 32.1 77.2 96.7 -- 2
40.3 83.3 100 -- 3 41.2 68.1 91.0 -- 4 24.0 50.5 65.9 87.1 5 44.2
71.0 91.2 -- 6 21.0 47.1 58.0 62.7 7 21.7 60.2 71.3 94.2 8 29.6
52.9 79.9 93.3 9 28.4 58.2 68.0 82.0 10 13.9 21.2 45.4 68.2 11 27.0
46.8 71.2 81.8 12 37.5 47.5 62.7 86.7 13 32.1 56.6 80.1 97.2
[0138] The data in Table 2 indicated that the sustained-release
pharmaceutical formulations prepared with the process of the
present application have a good sustained-release effect for
various types of active ingredients.
Example 5
[0139] Following the same procedure as in Example 1, using
leuprorelin acetate as an active ingredient, and using different
amounts of aluminium stearate, sustained-release pharmaceutical
formulations were prepared and the in vitro accumulated release
thereof were determined and listed in Tables 3 and 4.
TABLE-US-00006 TABLE 3 Sustained-release formulations of
leuprorelin acetate Amount of Amount of Nos. leuprorelin acetate
aluminium stearate 1 2 mg -- 2 1 mg 20 mg 3 1 mg 50 mg
TABLE-US-00007 TABLE 4 Results of the in vitro accumulated release
of the sustained-release formulations of leuprorelin acetate
Accumulated release of drugs % Nos. 1 d 3 d 5 d 7 d 1 53.4 69.7
84.1 99.8 2 20.6 37.4 76.0 94.0 3 17.2 41.6 76.7 100.0
[0140] Adding a small amount of aluminium stearate in the
formulations would improve the release performance of the drug, but
the release performance of the drug would reduce when an amount of
aluminium stearate is too high.
Example 6
[0141] The effects of the preparation manner on the in vitro
sustained-release performance of leuprorelin acetate were
investigated.
[0142] Without the preparation procedure, an amphipathic molecule
(e.g. EPC, Span 85) was directly added into an oily solvent,
followed by adding an active drug, dispersed uniformly, and the in
vitro accumulated release of the drug was determined with the
method described above. The results demonstrated that more than 90%
of the active drugs were released within 1 day. The
sustained-release performance was significantly lower than that of
the formulation obtained through the preparation process of the
present application. The results were shown in Table 5.
TABLE-US-00008 TABLE 5 Amphipathic Leuprorelin acetate molecules
and amount Accumulated Nos. and amount thereof thereof Preparation
manner release % 1 2.1 mg EPC 20 mg Direct dissolving 98.9
Cholesterol 5 mg without preparation (24 h) procedure 2 2.2 mg EPC
20 mg with preparation 53.4 Cholesterol 5 mg procedure (24 h) 3 1.8
mg Span 85 100 mg Direct dissolving 100 Cholesterol 5 mg without
preparation (12 h) procedure 4 2.1 mg Span 85 100 mg with
preparation 36.2 Cholesterol 5 mg procedure (24 h)
[0143] The results demonstrated that the formulations obtained
through the preparation process of the present application showed
better stability and better sustained-release performance.
Example 7
[0144] The effects of sustained-release formulations of calcitonin
on bone mineral density in ovariectomized rats were
investigated.
[0145] The animals used were wiste female rats weighed 160-200 g.
The instrument used was Bone Densitometer (LUNAR) manufactured by
General Electric Company, USA.
[0146] Experimental procedure: the rats were anesthetized by
intramuscular injection of saiantong anaesthesia compound formula
(10 mg/kg), shaved at the abdominal region, and cut alone the
middle line of the lower abdomen. Ovaries at both sides of the rats
were dissociated and excised. The muscles and skins at the
abdominal region were then sutured. Penicillin was intramuscularly
injected after the operation at 2 times per day for 3 days. On day
3 after the operation, formulation 12 as prepared in Example 4 was
administered once by subcutaneous injection at dosage of 8
.mu.g/kg. The bone mineral densities in the lumbar vertebra in the
third and forth week after the administration were determined,
respectively, and were compared with the pseudo-operation group in
which the ovaries were not excised from the rats and the model
group in which the ovaries were excised from the rats but no
calcitonin formulation was injected.
[0147] The results demonstrated that comparing with the model
group, the bone mineral densities in the lumbar vertebra of the
rats significantly increased in the third and Forth week after the
administration (p<0.05).
TABLE-US-00009 TABLE 6 Effects of sustained-release formulations of
calcitonin on bone mineral density in ovariectomized rats bone
mineral densities in lumbar vertebra after Dosage administration
(g/cm.sup.2) Groups (.mu.g/kg) Third week Fourth week
Pseudo-operation group -- 0.225 .+-. 0.157 0.232 .+-. 0.112 Model
group -- 0.195 .+-. 0.011.sup.= 0.197 .+-. 0.013.sup.==
Sustained-release 8.0 0.211 .+-. 0.011* 0.213 .+-. 0.018*
formulation of calcitonin Notes: .sup.=p < 0.05, .sup.==p <
0.01, comparing with the pseudo-operation group: *p < 0.05,
comparing with the model group; n = 8
Example 8
[0148] The effects of sustained-release formulations of exenatide
on blood glucose levels of mice were investigated.
[0149] Following similar procedure as in Example 1, using exenatide
as an active ingredient, injectable water as an aqueous phase
solvent, dichloromethane as an organic phase solvent, and using the
components as listed in Table 7, sustained-release formulations of
exenatide were prepared.
TABLE-US-00010 TABLE 7 Sustained-release formulations of exenatide
Eggyolk Injectable phosphatidyl Aluminium medium-chain Nos.
Exenatide choline Cholesterol stearate oil Formulation 1 5 mg 20 mg
10 mg -- 1 g Formulation 2 5 mg 20 mg 10 mg 20 mg 1 g Formulation 3
5 mg 20 mg 10 mg 50 mg 1 g
[0150] The animals used were male KK-Ay mice, aged 8-10 weeks and
raised under the conditions which complied with corresponding
standards.
Grouping and Drug Treatment of Animals
[0151] KK-Ay mice were randomly grouped into solvent control group,
positive control group, formulation 1 group, formulation 2 group,
and formulation 3 group according to weights and blood glucose
values, with 5 animals in each group.
[0152] Solvent control group: injectable medium-chain oil was
administered by intramuscular injection with a single dosage of 100
.mu.l per animal, 50 .mu.l at each hind leg;
[0153] Positive control group: a solution of exenatide in PBS
buffer was administered subcutaneously at the neck region with a
dosage of 0.06 .mu.g/100 .mu.l at 5:30 pm each day;
[0154] Formulation 1 group, formulation 2 group, and formulation 3
group: formulations 1, 2 and 3 in the above table were administered
by intramuscular injection, respectively, at a single dosage of 100
.mu.l per animal, 50 .mu.l at each hind leg.
[0155] Changes in the blood glucose level were monitored at
8:30-9:00 am each day. On the eighth day after administration, an
additional administration was performed; 4 .mu.g/100 .mu.l was
administered to each animal in formulation 1 group, formulation 2
group, and formulation 3 group; and 0.6 .mu.g/100 .mu.l was
administered to each animal in positive control group. Starting
from the ninth day, 0.18 .mu.g/100 .mu.l was administered to each
animal in positive control group twice a day.
[0156] The blood glucose level's of KK-Ay mice after starving for 4
h on 17th to 41st day after administration (Table 8) and after
starving for 12 h on 35th day after administration (Table 9) were
determined. The experiment results showed that all of the
formulations 1, 2 and 3 demonstrated significant and sustained
effects of reducing blood glucose in KK-Ay mice after starving for
4 h or 12 h, which may last for 24 to 27 days after administration,
in which the effects of formulation 2 are more significant.
TABLE-US-00011 TABLE 8 Blood glucose levels of KK-Ay mice after
starving for 4 h on 17th to 41st day after administration Blood
Glucose (mmol/L) Time Solvent control Positive control Formulation
1 Formulation 2 Formulation 3 (day) group group group group group
17 14.88 .+-. 4.10 11.5 .+-. 3.47 10.98 .+-. 3.15 10.72 .+-. 2.24
11.64 .+-. 3.04 22 23.56 .+-. 2.50 16.18 .+-. 3.98 18.72 .+-. 4.28
16.92 .+-. 3.03 18.04 .+-. 3.96* 27 19.88 .+-. 3.36 14.06 .+-. 3.58
16.90 .+-. 5.03 14.54 .+-. 3.53 16.28 .+-. 3.27 32 17.54 .+-. 3.51
12.56 .+-. 2.68 14.60 .+-. 2.65 15.14 .+-. 0.87 15.40 .+-. 6.07
TABLE-US-00012 TABLE 9 Blood glucose levels of KK-Ay mice after
starving for 12 h on 35th day after administration Blood Glucose
Groups (mmol/L) Solvent control group 10.3 .+-. 2.23 Positive
control group 6.16 .+-. 1.59** Formulation 1 group 6.58 .+-. 1.13*
Formulation 2 Group 6.84 .+-. 1.26* Formulation 3 group 7.34 .+-.
1.10* *p < 0.05, **p < 0.01, comparing with control group
[0157] In addition, formulations 2 and 3 showed significant effects
of suppressing food intake on the first day of administration
(specific to this drug). During the whole process of the
experiment, formulation 2 showed effects of suppressing food intake
which are similar to the positive control drug.
Example 9
[0158] In vitro release and in vivo efficacy of sustained-release
formulations of insulin were investigated.
[0159] Following similar procedure as in Example 1, using the
amounts of solvents as listed in Table 10, sustained-release
formulations of insulin were prepared from 5.0 mg of insulin, 20 mg
of eggyolk phosphatidyl choline, 10 mg of cholesterol, 20 mg of
aluminium stearate and 1 g of injectable medium-chain oil, and the
in vitro accumulated release of the formulations was determined
(Table 11).
TABLE-US-00013 TABLE 10 Sustained-release formulations of insulin
Aqueous phase solvents and amounts Organic solvents and amounts
Formulations thereof thereof 1 Aqueous solution of acetic acid 5 mL
Dichloromethane 20 mL 2 Aqueous solution of acetic acid 5 mL
Dichloromethane 10 mL 3 Aqueous solution of acetic acid 0.5 mL
Dichloromethane 20 mL 4 Aqueous solution of acetic acid 0.5 mL
Dichloromethane 9.5 mL Acetone 0.5 mL 5 Aqueous solution of acetic
acid 5 mL Dichloromethane 19.5 mL Tert-butanol 0.5 ml 6 Aqueous
solution of acetic acid 5 mL Dichloromethane 19.5 mL Ethanol 0.5 mL
7 Aqueous solution of acetic acid 5 mL Dichloromethane 19.5 mL
Isopropanol 0.5 mL 8 Aqueous solution of acetic acid 5 mL
Dichloromethane 9.5 mL Tert-butanol 1.0 mL
TABLE-US-00014 TABLE 11 In vitro accumulated release of
sustained-release formulations or insulin Accumulated release of
drug % Formulations 1 day 3 days 5 days 9 days 1 3.75 10.0 40.1
57.3 2 13.2 27.2 47.9 100 3 6.31 17.6 69.3 97.5 4 24.1 33.8 55.8
92.3 5 4.66 14.1 31.8 45.9 6 19.6 26.2 64.1 100 7 21.1 46.7 77.0
100 8 18.0 32.3 76.5 96.3
[0160] The data in Table 11 demonstrated that the testing
formulations slowly released the drug in vitro in at least 9
days.
In Vivo Efficacy of Sustained-Release Formulations of Insulin
[0161] Experimental procedure: basically following the previously
disclosed process (Lijiang Song, et al., Observation of effect of a
glucose-reducing medical care capsule on model mice, Journal of
Chinese Medicine Research, 6(1):53-55, 2006). After starved and fed
by only water for 24 hrs. mice were intraperitoneally injected with
160 mg/kg of streptozotocin. After 72 hrs, blood glucose levels at
fasting for 6 h were determined. Mice with blood glucose of 15-30
mmol/L were classified as qualified for the model and were evenly
divided into different groups. On the fourth day, mice were
injected with the insulin formulations 1-8, respectively, while the
control group was only given the auxiliaries. After injection,
blood glucose levels were determined at fasting for 6 hrs at 3 h,
24 h, 3 d, 5 d, 7 d and 9 d, respectively. One drop of blood was
taken from each animal by cutting tails thereof, and was added onto
a OneTouch.RTM. Basic.RTM. Blood Glucose Meter glucose oxidase Test
Strip manufactured by LifeScan Inc. of Johnson & Johnson Ltd.
to measure blood glucose levels. The results were listed in Tables
12-13 below.
TABLE-US-00015 TABLE 12 Efficacy of sustained-release formulations
of insulin in treating mice having streptozotocin-induced diabetes
Blood glucose (mmol/L) Dosage 3 hrs after Groups (U/kg) 0 day
administration 1 day 5 days 9 days 14 days Control 26.6 .+-. 2.1
26.3 .+-. 2.0 21.8 .+-. 5.7 19.3 .+-. 3.9 20.0 .+-. 3.5 21.6 .+-.
3.9 Formulation 10 26.5 .+-. 1.8 22.6 .+-. 2.4 7.1 .+-. 4.8* 11.8
.+-. 4.1* 12.2 .+-. 1.1* 22.1 .+-. 2.2 1 30 27.5 .+-. 1.8 21.1 .+-.
2.2 3.0 .+-. 1.1* 9.6 .+-. 5.4* 12.6 .+-. 0.5* 19.2 .+-. 2.3 *P
< 0.05
[0162] It can be seed from the results listed in Table 12 that for
insulin formulation 1 which was subcutaneous injected at 10 U/kg
and 30 U/kg, the peak of the effects of reducing blood glucose is
at 24 hrs, and the effects lasted for 9-14 days.
TABLE-US-00016 TABLE 13 Efficacy of different sustained-release
formulations of insulin in treating mice having
streptozotocin-induced diabetes Dosage Blood glucose (mmol/L)
Groups (U/kg) 0 day 1 day 3 days 5 days 7 days 9 days Control 19.1
.+-. 2.5 18.3 .+-. 4.4 22.4 .+-. 4.3 20.6 .+-. 4.2 21.9 .+-. 3.1
22.7 .+-. 4.2 group Formulation 10 19.1 .+-. 2.5 5.5 .+-. 1.0* 7.1
.+-. 4.8* 8.3 .+-. 1.2* 11.8 .+-. 4.1* 22.7 .+-. 4.2 5 30 19.1 .+-.
2.5 2.6 .+-. 0.6* 3.0 .+-. 1.1* 6.1 .+-. 4.0* 9.6 .+-. 5.4* 21.5
.+-. 3.2 60 19.0 .+-. 2.4 1.9 .+-. 0.8* 2.4 .+-. 1.0* 6.4 .+-. 6.2*
13.5 .+-. 5.5* 16.4 .+-. 6.4 Formulation 10 21.4 .+-. 3.5 8.0 .+-.
4.1* 15.4 .+-. 5.0* 21.5 .+-. 2.6 21.2 .+-. 1.6 24.0 .+-. 3.2 2 30
20.3 .+-. 1.9 2.8 .+-. 0.9* 3.3 .+-. 1.5* 18.3 .+-. 8.8 17.8 .+-.
6.1 22.4 .+-. 4.7 60 20.5 .+-. 2.1 3.1 .+-. 2.4* 9.1 .+-. 8.2* 22.0
.+-. 0.8 20.9 .+-. 0.6 21.6 .+-. 3.0 Formulation 30 22.4 .+-. 1.2
5.7 .+-. 2.7* 19.0 .+-. 3.03 22.8 .+-. 1.6 19.7 .+-. 2.7 22.8 .+-.
3.9 3 *P < 0.05. comparing with control group
[0163] It can be seen from the results listed in Table 13 that the
significant effects of formulation 3 in reducing glucose lasted for
1 day, the significant effects of formulation 2 in reducing glucose
lasted for 3 days, and the significant effects of formulation 5 in
reducing glucose lasted for 7 days.
[0164] A person skilled in the art would understand that the term
"such as" or "for example" as used herein represents "including,
but not limited to".
[0165] Although the present application is described through the
above embodiments and specific descriptions, but the present
applicant is not limited thereto. In view of the disclosure of the
present application, a person skilled in the art may make
modifications or changes the technical features in the above
embodiments without departure from the spirit of the present
application, and these modifications or changes are within the
scope of the present application.
* * * * *